Ozone stress response of leaf BVOC emission and photosynthesis in mountain birch (Betula pubescens spp. czerepanovii) depends on leaf age

Abstract Oxidative stress from ozone (O3) causes plants to alter their emission of biogenic volatile organic compounds (BVOC) and their photosynthetic rate. Stress reactions from O3 on birch trees can result in prohibited plant growth and lead to increased BVOC emission rates as well as changes in their compound blend to emit more monoterpenes (MT) and sesquiterpenes (SQT). BVOCs take part in atmospheric reactions such as enhancing the production of secondary organic aerosols (SOA). As the compound blend and emission rate change with O3 stress, this can influence the atmospheric conditions by affecting the production of SOA. Studying the stress responses of plants provides important information on how these reactions might change, which is vital to making better predictions of the future climate. In this study, measurements were taken to find out how the leaves of mature mountain birch trees (Betula pubescens ssp. czerepanovii) respond to different levels of elevated O3 exposure in situ depending on leaf age. We found that leaves from both early and late summers responded with induced SQT emission after exposure to 120 ppb O3. Early leaves were, however, more sensitive to increased O3 concentrations, with enhanced emission of green leaf volatiles (GLV) and tendencies of both induced leaf senescence as well as poor recovery in the photosynthetic rate between exposures. Late leaves had more stable photosynthetic rates throughout the experiment and responded less to exposure at different O3 levels.

. Since BVOCs are produced through light-and temperature-dependent processes, they are affected by changes in these factors as well as by additional abiotic and biotic factors causing plant stress.As a defense against stresses, plants respond by altering their constitutive BVOC emissions, either by changing their compound composition or even by inducing emissions of compounds they have not emitted before (Holopainen & Gershenzon, 2010;Loreto & Schnitzler, 2010;Sharkey et al., 2008).Once emitted, either constitutively or induced by plant stress, BVOCs are part of several chemical chain reactions in the atmosphere where terpenoids like isoprene, monoterpenes (MTs), and sesquiterpenes (SQT) are highly reactive.The emitted compounds become part of gas-phase oxidative reactions with air pollutants and greenhouse gases like tropospheric ozone (O 3 ) that are formed by photochemical reactions in the presence of nitrogen oxides (NO x ).The gas-phase reactions from the BVOCs are affecting the properties of the atmosphere by enhancing the production of secondary organic aerosols (SOA; Laothawornkitkul et al., 2009;Monson & Baldocchi, 2014), leading to changes in the energy budget of the earth by scattering the incoming radiation.
Oxidation products from BVOC can also act as cloud condensation nuclei, enhancing cloud formation and affecting the energy budget similarly to SOA (Monson & Baldocchi, 2014;Paasonen et al., 2013;Scott et al., 2018).A shift in the emission of BVOCs can thus have large implications for atmospheric chemistry and radiative forcing.
Not only do induced BVOC emissions impact the atmosphere, but they also have high metabolic costs for the plants themselves (Keeling & Bohlmann, 2006).The high metabolic costs are evident from plant stress from elevated chronic tropospheric O 3 , resulting in degrading crop yields and decreased forest growth from losses in photosynthetic carbon assimilation (Loreto & Schnitzler, 2010;Mills et al., 2011).This aligns with responses seen in BVOC emissions to abiotic stress from O 3 exposure, where, depending on the plant species and its O 3 tolerance, emissions were both induced or reduced (Esposito et al., 2016;Kask et al., 2021;Peron et al., 2021;Vitale et al., 2008).Exposure to O 3 for extended periods also led to changes in the BVOC blend, mainly with induced MT emissions (Loreto et al., 2004;Moura et al., 2022;Rinnan et al., 2005).Induced emissions of SQT were also found to be positively correlated with increases in O 3 concentration (Bourtsoukidis et al., 2012;Moura et al., 2022).Apart from chronic O 3 exposure, it is also important to consider short-term ozone exposure at high doses (acute stress) that can cause major harm to plants by inducing cell death and altering metabolic profiles without visual damage (Vainonen & Kangasjärvi, 2015).Some tree species are more tolerant to O 3 , often related to leaf glandular trichomes that have been proven to protect the plants against O 3 stress by exuding metabolites that deplete O 3 , where the level of protection is correlated with the density of the trichomes (Li et al., 2018;Mofikoya et al., 2018;Valkama et al., 2004).
Leaf age has also been proven to be important for O 3 protection.
Younger leaves tend to have better antioxidant protection against O 3 damage compared to older leaves in terms of foliar injury and increases in metabolite concentration (Wedow et al., 2021;Zhang et al., 2010).
Leaf age also matters in terms of BVOC emissions; higher constitutive emissions were found from young leaves of downy birch (Betula pubescens) compared to older leaves (Hellén et al., 2021).In general, the BVOC emission profile from birch trees is dominated by MT and SQT with little contribution from isoprene (Haapanala et al., 2009;Hakola et al., 2001;Hellén et al., 2021).The BVOC emission profile of birch was, however, altered by O 3 exposure; potted birch saplings increased their overall emission rates when exposed to concentrations >80 ppb for long periods (Carriero et al., 2016), while MT emissions were decreasing with 150 ppb short-term exposure (Timkovsky et al., 2014).Birch trees are considered an O 3 -sensitive species where high exposure has affected tree growth negatively and promoted leaf senescence (Karlsson et al., 2003;Maurer & Matyssek, 1997;Saleem et al., 2001).The sub-Arctic mountain birch (Betula pubescens ssp.czerepanovii) can, in particular, be further sensitive as the protective glandular trichomes on mountain birch leaves have a lower density compared to other common birch subspecies (Valkama et al., 2004).
Given the impact that BVOCs have on the chemistry and physics of the atmosphere and the resulting effects on climate change, it is crucial to understand how elevated O 3 exposure affects leaves, especially in areas where the leaves have not adapted to high O 3 levels yet.Mountain birch grows in the sub-Arctic part of Sweden, an area not experiencing as high O 3 exposures as the rest of northern Europe (Klingberg et al., 2019).The Arctic air quality is, however, expected to be degraded by increased ship traffic, causing increases in O 3 concentrations of approximately 5% by 2030 (Gong et al., 2018).This makes it a relevant area for studying The field site was visited twice, during the start of summer (22nd of June-3rd of July) and during the peak growing season (27th of July-5th of August), herein referred to as early and late summer (Table 1).
All trees in the area had started to develop leaves about 2 weeks before the first field visit (8th of June; ANS, 2023).The leaves were fully unfolded when the measurement started.The area has a history of autumnal moth (Epirrita autumna) outbreaks every 10-11 years that strongly modify the BVOC emissions of mountain birch when numerous larvae eat the leaves (Rieksta et al., 2020).There was no indication of an outbreak during the measurement year and the latest outbreak occurred in 2012/2013 (Olsson et al., 2017;Tenow, 1996), meaning that the measured emission rates during the study were not influenced by herbivory.
The long-term mean air temperature (year 1991-2020) during the growing season (June to August) in Abisko ranges from 9°C to 12°C with a precipitation sum of 140 mm (SMHI, 2023).In 2020, the growing season was a bit warmer than the long-term average (15 ± 3°C (mean ± standard deviation)), while the precipitation was similar to the long-term average with 143 mm.The daily amplitude in temperature was large; the lowest and highest temperatures during the first field visit were 1°C and 21°C, while it was slightly warmer during the later field visit, with the lowest and highest temperatures being 6°C and 22°C (Figure 1).The sum of precipitation during the field visits was 5 mm and 1 mm, respectively.
Three mature mountain birch trees growing within 4 meters of each other were selected for repeated measurements during the study period (Table 1).The trees were at least 40 years old and between 3 and 4 m tall.One branch from each tree was selected for the study; the branches on birch 1 and 2 were at similar heights of around 1.40 m, and the branch on birch 3 was located further down the tree at around 0.7 m.Leaves in direct sunlight were excluded from the selection.The same branches were measured during both field visits.

| Experimental design
The measurement setup consisted of a pump system and a port- From the same air stream, BVOC samples were taken by extracting air through adsorbent tubes using a pocket pump (SKC Ltd., Dorset, UK).During the sampling period, there was an automatic toggling three-way valve installed to swap between air before and after the chamber with 2-min intervals to monitor the different O 3 levels and potential degradation through the photosynthesis system to know the approximate concentration inside the leaf chamber.
Each leaf was measured at all O 3 concentration steps following a measurement sequence starting at 0 ppb (control) for 30 min, followed by an exposure phase of 40 ppb for an hour, back to 0 ppb for 30 min again (recovery phase 1), an exposure phase of 80 ppb for an hour, 0 ppb for 30 min (recovery phase 2), 120 ppb for an hour, and, lastly, 0 ppb for 30 min (recovery phase 3; Figure 2b).Each leaf was sampled for BVOC seven times following the sequence (Figure 2b), resulting in a total of 147 samples (Table 1).After each measurement sequence, the leaves were harvested and photographed for specific leaf area (SLA) calculations.They were weighted and dried TA B L E 1 Sampling conducted during 2020 at the field site in Abisko (northern Sweden) for the field visits early and late in the summer.Note: The same three birch trees and branches were measured during both field visits, and the number of leaves measured each time along with the number of samples and the total amount of leaves and samples were summed for each field visit and the entire study.
at 70°C until the dry weight was stable.Additional measurements of chlorophyll content were taken on five leaves on the same branch using a chlorophyll meter (SPAD-502 Plus, Konica Minolta Sensing, Inc., Japan).

| BVOC sampling
Before starting the measurements, the leaves were inserted into the chamber of the LI-6400 with a flow rate of 750 μmol s −1 and al- To identify possible background contamination, background BVOC concentrations were sampled through similar adsorbent tubes and pocket pumps at two locations in the experimental setup: after the buffer volume and directly after the chamber with no leaf inside (Figure 2a).The background samples were done twice a day and sampled for 30 min with a flow rate of 200 mL min −1 .After collecting the samples, all tubes were capped with long-term storage caps and stored in a refrigerator (~3°C) before analysis.
The method has been described in Helin et al. (2020).
The leaf dry weight (g dw ) inside the chamber was calculated according to Equation (1): where g dw tot is the dry leaf weight of the whole leaf (g), LA is the total leaf area (cm 2 ), and CA is the leaf area inside the chamber, which was always 6 cm 2 .
As PAR and leaf temperature were set to constant values, the emission rates were not standardized but reported as measured emission rates at 20°C and 1000 μmol m −2 s −1 PAR.The compound blend (%) was calculated as the emission of the emitted compounds divided by the sum of the emissions of all identified BVOCs.The ratio between compound groups were calculated similarly.The detection limit (LOD) was calculated for each compound as emission rates based on blank measurements resulting in LOD ranging from 0.45 to 10 ng g dw −1 h −1 (see Table S2 for LOD for each compound).
SLA (cm 2 g −1 ) was calculated as LA divided by g dw tot .

| Statistical analysis
Our data sets were tested for normality using the Kolmogorov-Smirnov test and creating normal probability plots (kstest, (1) Schematic of the (a) instrumental set-up and (b) measurement sequence.Ambient air entered the system from three inlets, one through a VOC filter and one through an O 3 generator, which were later joined in a buffer volume to deliver O 3 -enriched air to the system.The third air inlet was located directly at the inlet of the photosynthesis system (LI6400) and included a scrubber for VOCs and O 3 to use as O 3 -free air during the 0 ppb measurements.During the O 3 exposure measurements, the O 3 -enriched air was directed to an O 3 monitor and the LI6400.Air was sampled through adsorbent tubes using pocket pumps from two places: after the chamber head (for leaf emissions) and after the buffer volume (to get background emissions).Using an automatic toggling three-way valve, the O 3 monitor was receiving air before the LI6400 and after the chamber head to see concentrations before and after the chamber.One leaf was sampled for 30 min at 0 ppb before O 3 exposure, after which it was exposed to 40 ppb, 80 ppb, and 120 ppb for 60 min each, with a recovery phase of 30 min in-between.
Samples were taken at each step in the sequence, resulting in seven samples in total for one leaf.(mean ± standard deviation) for the total BVOC compounds, while the late leaves emission rate was higher with an average of 456 ± 876 ng h −1 (mean ± standard deviation; Figure 3; Table S2).Separated into compound groups, early leaf MT emission was 100 ± 170 ng h −1 and late leaf MT emission was higher at 433 ± 882 ng g dw Oxygenated compounds (OXY), SQT, and isoprene emissions were h −1 ).The early leaf photosynthetic rate was on average 11 ± 5 μmol m −2 s −1 and 14 ± 2 μmol m −2 s −1 for the late leaves (Table S2), which shows a relatively higher fraction of resources spent in spring for OXY, SQT, and isoprene, but the opposite for MT.Despite the higher total emission rates for the late leaves, the difference was not significant.However, the emission rates for two individual compounds indicated a significant difference: camphene was significantly higher (p < .03)for the late leaves, and longicyclene was significantly lower (p < .003)for the late leaves compared to the early leaves (Table S2).
The photosynthetic rate was significantly higher for the late leaves (p < .03;Table S2), along with significant differences for SLA, chlorophyll content, transpiration rate, and stomatal conductance (p < .001for all), where SLA was higher for early leaves but chlorophyll content, transpiration rate, and stomatal conductance were lower compared to late leaves (Table S2).
We found high variabilities in BVOC emission rates between leaves from different branches and also among leaves from the same branch (Figure 3; Figure S2).The compound blend also varied much, especially for the early leaves, where half emitted mainly OXY compounds (Table S1).The emissions from late leaves were mainly dominated by α-pinene for all but two leaves (Table S1).S3).The emission blend for late leaves was similar to the control, mainly the emission of α-pinene for all birch branches and recovery phases (Figure S3).However, the recovery phases for the early leaves varied among the birches, with main increases in compound contribution (% of total emissions) for linalool (50%), isoprene (20%), MBO (15%), and cis-3-hexenol (8%) on average for Birch 1 and 2. Birch 3, on the other hand, had the largest increase both in emission rate and emission contribution, dominated by α-pinene with 87% during the third recovery phase (Figure 4; Figure S3).

| BVOC emission patterns and photosynthetic rate during the recovery phase
Similar photosynthetic rates were observed for early leaves between the birches during the control state and the first recovery phase (10-12 μmol m −2 s −1 ; Figures 3 and 4).Photosynthetic rates did, however, decrease by 3 μmol m −2 s −1 for all birches during the second recovery phase compared to the first (Figure 4).However, during the third recovery, they responded differently in photosynthetic rate: Birch 1 increased to similar rates as the control and first recovery phase (11 μmol m −2 s −1 ), Birch 2 continued to decrease in photosynthetic rate to 7 μmol m −2 s −1 , and Birch 3 showed no difference from the second recovery phase (Figure 4).The late leaves instead have a larger difference in photosynthetic rate between the birches in the control state, 11, 13, and 15 μmol m −2 s −1 for Birches 1, 2, and 3, respectively.However, the photosynthetic rate instead remained stable throughout all recovery phases for all birches (Figure 4).

| BVOC emission patterns and photosynthetic rate during O 3 exposure
To investigate the effects of O 3 exposure on leaf age and find a potential stress threshold, the exposure phases of the measurement sequence included step-wise increases in the O 3 concentration, which can be considered a light (40 ppb), medium (80 ppb), and severe (120 ppb) O 3 stress level.We found that, similar to the control and recovery stages, the emission blend profile varied considerably for both early and late leaves (Figure S4).The emission rates depending on leaf age indicated differences during the different exposure stages.Total emission rates were 2-50 times higher from early leaves compared to late leaves during the same exposures of 40 ppb and 120 ppb (Figure 5; Table S4).The differences between early and late leaves were slightly smaller at 80 ppb, but emission rates were still 2 times higher for the early leaves (Figure 5; Table S4).
The early leaves from Birches 1 and 2 had similar patterns where their emission blend indicated a 95% increase of cis-3-hexenol when exposed to 40 ppb compared to the control, while Birch 3 was deviating from the pattern with an 80% increase of α-pinene instead (Figure S4).Compared to the control, the early leaves from Birch 1 and 3 decreased the total emission rates with 112 and 68 ng g dw −1 h −1 at 40 ppb, respectively, while Birch 2 instead increased emission rates with 129 ng g dw −1 h −1 .At 80 ppb, the compound blend of Birch 2 was dominated by linalool (70%) and Birch 3 by isoprene (70%), while Birch 1 had limonene (30%) and α-pinene (20%) as main contributors (Figure S4).Emission rates were 80% lower for all birches at 80 ppb compared to 40 ppb and control (Figure 5; Table S4).Exposure at 120 ppb revealed no large change in emission rates or compound blend for Birch 2, other than small (>8%) compound blend increases in cis-3-hexenol and MBO compared to 80 ppb (Figure S4; Figure 5; Table S4).Exposure at 120 ppb for Birches 1 and 3 had larger implications in their blend.SQTs α-humulene, β-caryophyllene, and βfarnesene dominated the emissions (90%) from Birch 1 that also had the highest emission rates (560 ng g dw −1 h −1 ) of all stages (Figure S4; Figure 5; Table S4).Birch 3 mainly emitted MBO, linalool, isoprene and β-farnesene (together 77% of their emissions), with emission rates remaining similar to the exposure at 80 ppb (Figure S4; Figure 5; Table S4).The photosynthetic rates were seen to decrease on average by 15% and 19% at each step for Birches 1 and 2, respectively, compared to the control stage, while they were stable for Birch 3 until the last exposure stage (120 ppb) where they decreased (Figure 5; Table S4).
When the late leaves were exposed to 40 ppb, all emissions changed from α-pinene dominance (40%-80% of total emissions) to a higher variety of mainly MBO, limonene, isoprene, and β-caryophyllene (Figure S4).The emission rates for all control stage late leaves were 40 times higher than the late leaf emission rates at 40 ppb (Table S4).

| The average BVOC emission and photosynthetic rate for all measurement sequence steps
An average of the BVOC emission and photosynthetic rate was calculated for early and late leaves.The average followed similar patterns given by the black whiskers.The BVOC compounds are separated by color in their respective groups, with monoterpenes (MT) in red-pink, sesquiterpenes (SQT) in greens, and oxygenated compounds (OXY) in blues, where MBO is abbreviated from 2-methyl-3-buten-1-ol and AMCH from 4-acetyl-1-methylcyclohexene.Isoprene is separated on its own and colored yellow.
(16 to 73 ng g dw −1 h −1 ; Table S4; Figure 6a).The early leaves had non-significantly lower emission rates compared to the control at recovery 1, 2, and 80 ppb, but the emission rates non-significantly increased with 139 ng g dw −1 h −1 at 120 ppb and changed in blend to mainly SQTs (80%; Figure 6a).At recovery 3, the early leaf emission rate non-significantly increased 5 times compared to the control state, where α-pinene contributed 80% to the increase (Figure 6a).
The late leaves had a more obvious pattern, with significantly lower (p < .02)total emission rates at all exposure stages compared to the control and recovery stages (Figure 6b; Figure 7).The recovery stages also indicated a steady but non-significant decrease in emission rate after each exposure, where the emissions decreased on average by 20% after exposure compared to the control stage (Figure 6b).
The photosynthetic rate showed tendencies to decrease at each step compared to the control state, irrespective of leaf age, where the decrease from control to last recovery was 2 μmol m −2 s −1 for early leaves and 1.5 μmol m −2 s −1 for late leaves (Figure 6a; Table S3; Table S4).Stomatal conductance for early and late leaves followed similar patterns, where the main difference between them was the magnitude, as the late leaves had higher stomatal conductance compared to the early leaves (10 mol H 2 O m −2 s −1 higher; Figure S1).After exposure at 80 ppb, there was always a significant decrease in the early leaf photosynthetic rate compared to the control leaves at 40 ppb and recovery 1 (p < .001; Figure S5).The stomatal conductance of the early leaves was significantly lower during exposure to 80 ppb compared to the control (p < .001; Figure S5).Despite this, there was no significant difference in the total emission rates or the compounds for the early leaves (Figure S5).The late leaves, however, had a significantly lower photosynthetic rate already after 40 ppb compared to the control (p < .03),but only significantly lower stomatal conductance after exposure to 120 ppb compared to the control (p < .001; Figure S6).Late leaves also had significantly lower emissions of α-pinene (p < .02)when the leaves were exposed to O 3 (Figure 7).There were also small differences for the compounds βcaryophyllene, p-cymene, 3Δ-carene, and camphene, which had the main differences between the control state and the exposure phases The standard deviation (SD) from the mean for the BVOC emission rates is given by the black whiskers.The BVOC compounds are separated by color in their respective groups with monoterpenes (MT) in red-pink, sesquiterpenes (SQT) in greens, and oxygenated compounds (OXY) in blues, where MBO is abbreviated from 2-methyl-3-buten-1-ol and AMCH from 4-acetyl-1-methylcyclohexene.Isoprene is separated on its own and colored yellow.

| Changes in the ratio of monoterpenes to sesquiterpenes depending on exposure and recovery
As a further stress indicator, the ratio between MT and SQT was relevant due to their different production pathways.When comparing the sum of the MT and SQT for early leaves, we saw that MT dominated all steps except at 120 ppb, where SQT was dominating at almost 100%, but the opposite was seen at recovery 3 (Figure 8a).
For the late leaves, there was a clear division between the ratios for the control and recovery stages compared to the O 3 exposure stages: MT was always dominating when there was 0 ppb O 3 , while SQT was dominating during the exposure stages (Figure 8b).

| Constitutive mountain birch BVOC emissions and photosynthetic rate depending on age and variation between leaves
Leaves in this study measured without prior exposure to O 3 represent the constitutive BVOC emissions from mountain birch (Betula pubescens ssp.czerepanovii).We found emission rates to depend on leaf age; early leaves emitted more SQTs and OXYs compared to late leaves, which instead emitted more MT.The age-dependent differences in this study were not statistically significant, but they were in agreement with the study by Mofikoya et al. (2018), which found similar, significant differences.Early leaves in our study tended to have higher variability in emission profiles and were mainly dominated by linalool, limonene, α-pinene, and β-farnesene, compared to late leaves that mainly emitted α-pinene (Table S1).The differences in emission profiles for leaf age were also found in Hellén et al. (2021), analyzing BVOC emissions from downy birch (Betula pubescens), finding early leaf emissions to be linalool-and SQT-dominated and late leaves dominated by MTs.Measurements of constitutive mountain birch emission rates have been reported previously, and Haapanala et al. ( 2009) and Ahlberg (2011) measured leaf emissions in the same area with similar timing and temperature (20°C) as our late leaves.
Our measured MT emissions are within the range from both studies (12 to 4000 ng g dw −1 h −1 ), while the SQT emissions in our study were only comparable to Ahlberg (2011) of 5.5 ng g dw −1 h −1 , as higher SQT emissions ranging between 25 and 2700 ng g dw −1 h −1 were found by Haapanala et al. (2009).The lower SQT emissions in this study could potentially be explained by the experimental setup.Since we installed MnO 2 nets before the sampling tubes to remove O 3 , there is a possibility that the SQT emissions are underestimated as these nets were found to scrub SQTs from the air (Hellén et al., 2023).
Expectedly, we also found early leaves to have lower and more variable photosynthetic rates compared to late leaves, which were both higher and more stable with respect to photosynthesis (Bielczynski The average BVOC emission and photosynthetic rate for each step in the measurement sequence (control state, exposed to 40 ppb, recovery 1 (0 ppb), exposed to 80 ppb, recovery 2, exposed to 120 ppb, recovery 3) for (a) early summer and (b) late summer.The standard deviation from the mean (SD) for the BVOC emission rates is given by the black whiskers.The BVOC compounds are separated by color in their respective groups, with monoterpenes (MT) in red-pink, sesquiterpenes (SQT) in greens, and oxygenated compounds (OXY) in blues, where MBO is abbreviated from 2-methyl-3-buten-1-ol and AMCH from 4-acetyl-1-methylcyclohexene.Isoprene is separated on its own and colored yellow.
et al., 2017).The photosynthetic rates for the leaves were also in line with the previous study by Ahlberg (2011).
In addition to our findings that photosynthetic and BVOC emission rates change with leaf age, we also found large variations in both photosynthetic and BVOC emission rates within both age groups.Variations between trees are expected regardless of age, depending on the different chemotypes (Bäck et al., 2012;Haapanala et al., 2009).Not commonly reported are variations from leaves on the same branch of a tree, which was the case for the leaves in this study.One part of the variation between the early leaves could potentially be explained by the high emissions of α-pinene from two leaves on Birch 3 (Figure S2a).A-pinene was the dominant compound and emitted at higher rates from late leaves compared to early leaves, however, not significantly (Table S2).Based on these results, we hypothesize that the high emissions of α-pinene from the two early leaves are an indication that they were more mature compared to the other early leaves.
To confirm this, we repeated the statistical analysis comparing leaf age emissions while excluding early leaves from Birch 3.This resulted in significantly higher α-pinene emissions in late summer compared to early leaves (p = .028).We do, however, not expect the varying maturity to contribute to the variations we saw for the late leaves; thus, another explanation could be sun exposure.BVOC emissions have been found to be affected by sunlit or shaded conditions (Karlsson et al., 2021;Keenan et al., 2011), and that could potentially explain the variations.During leaf selection, we did, however, take this into consideration and only chose leaves that were not directly sunlit upon measurements.
Leaves from the same branch were also always at the same height and from the outer part, suggesting that sun exposure variations between leaves are low.Another limitation of the study design is the sampling method.Adsorbent tubes give the accumulated The resulting p-values between the steps in the measurement sequence for both early and late summer photosynthetic rates, total emission rates, and from the compound α-pinene.A dark blue box indicates a significant difference (p < .05) between the control/ exposure/recovery phases.Statistical analysis was performed using a Kruskal-Wallis test followed by a multiple comparisons procedure with Dunn-Sidák's approach to investigate which groups were different from each other.
concentration during the measurement time, in our case for 1 h during O 3 exposure and 30 min when not exposed.This means that quick adjustments by the leaves cannot be seen with this method.
Other methods for quantifying rapid changes in BVOC emissions exist, such as a PTR-ToF-MS, and could facilitate fast adjustment measurements; however, using such instrumentation is limited in field studies similar to this one, and PTR-techniques are not able to separate compounds that share the same mass, i.e.MT compounds cannot be distinguished.To determine the cause of the within-branch leaf variability, more data and potentially different sampling methods would be needed.Our findings still highlight the importance of considering leaf-scale measurements in addition to branch-scale.

| Exposure to O 3 alters BVOC emissions depending on concentration level and leaf age
Trees have various ways of protection against oxidative stress from O 3 , for example, reducing stomatal conductance to avoid O 3 from entering the leaf, exuding metabolites from glandular trichomes, or inducing BVOCs to react with O 3 outside the leaf (Calfapietra et al., 2013;Li et al., 2018;Pääkkönen et al., 1996).When measuring the impact of O 3 in this study, we focused on the emission rates of BVOCs and the direct response of these.Other protection mechanisms were not studied, but as glandular trichome density for mountain birch does not change when leaves are fully unfolded to mature (Valkama et al., 2004), this should not influence the results in O 3 responses with leaf age.
When exposed to 40 ppb, early leaves did not change the total emission rate compared to the control, but we saw increases in emissions of the green leaf volatile (GLV) cis-3-hexenol (Figure 6a).
Cis-3-hexenol is emitted through the LOX pathway in the leaf, and products from this pathway have been proven to be induced by O 3 leaf damage (Li et al., 2018).We did not witness the same induction of cis-3-hexenol for late leaves at 40 ppb, but the emission rates were instead significantly lower at 40 ppb compared to the control, with an emission rate almost 20 times higher (Figure 6b).The significant response of the late leaves at 40 ppb was not expected since it is not uncommon that trees in Abisko experience similar ambient concentrations.Between 2004 and 2008, the maximum monthly O 3 concentrations were between 32 ppb and 49 ppb on average in Latnjajaure, close to Abisko (Klingberg et al., 2009).Because of this, our expectation was that emission rates for late leaves at 40 ppb would be similar to the control.The reason the results differ from our expectation can be due to the method where control leaves were exposed to O 3 -free air, common for chamber measurements of BVOCs (Ortega & Helmig, 2008).However, there is always some level of O 3 in the ambient air, and the constitutive control emissions we found might instead be an overestimation of the emission rates from the birch leaves outside the enclosures, at least for late leaves.
The largest response to acute O 3 exposure was from the early leaves on Birch 1 at 120 ppb O 3 (Figure 5).The other birches acted differently for early leaves; Birch 3 responded similar to the late leaves, with decreased emission rates at O 3 exposure and higher when not exposed.Birch 2 responded similarly to Birch 3, but had a different compound blend mainly dominated by SQT.We believed this to be due to differences in chemotype as previously found for mountain birch in Abisko (Haapanala et al., 2009); however, the same birch was not dominated by SQT later in the summer, indicating that this is not the case.This further highlights the variability of emission rates by age.Late leaves responded similarly to O 3 exposure, with emission rates at comparable levels at all exposures, and did induce emissions as much as early The emission rate ratio in percent (%) for (a) early summer and (b) late summer emissions of the group monoterpenes (MT) and the group sesquiterpenes (SQT).The ratio between the BVOC compound groups is displayed for each step of the measurement sequence (non-exposed, exposed to 40 ppb, 80 ppb, and 120 ppb and the respective recovery phases).
leaves (Table S4).The compound group increasing the most for both ages was SQT at 120 ppb (Figures 5 and 6).This is similar to Bourtsoukidis et al. (2012), who found O 3 stress to be a driver for SQT emissions from Norway spruce (Picea abies).In this study, the SQT emissions were mainly induced after exposure to 120 ppb but not after 80 ppb.Our findings thus suggest that a potential stress threshold lies between 80 and 120 ppb upon acute O 3 exposure.
Apart from SQT emission, cis-3-hexenol was already induced at 40 ppb for two of the birches during the early season; this indicates that early leaves might be more sensitive to O 3 at lower concentrations that might not be seen as SQT induction (Figure 5a).
Based on the results, looking at the ratio of SQT:MT might indicate O 3 stress, as an induction of SQT emission was seen with O 3 stress (Figures 5 and 6).From the ratio, the O 3 stress on early leaves through induced SQT is only seen at higher O 3 concentrations (120 ppb), while late leaves respond with increased SQT:MT at each exposure (Figure 8).This indicates a faster defense response from late leaves.The late leaves could respond faster due to lowconcentration priming effects from ambient O 3 concentrations (Li et al., 2017), as they have naturally been exposed to O 3 for longer.
This was also seen through photosynthesis, with steadier rates for late leaves throughout the experiment compared to early leaves.The early leaf photosynthesis rates also did not recover between the exposure stages (Table S3 and S4).The same was seen for the stomatal conductance, which indicated stress earlier for the early than the late leaves.This further suggests early leaves are more vulnerable to O 3 stress than late leaves.

| O 3 -induced BVOC emission implications for the sub-Arctic areas
The volatility and oxidative products of BVOCs determine the efficiency of growing SOA particles.Emitted compounds have different SOA yields; compound blend changes can then impact the locally produced SOA (Lee et al., 2006;Scott et al., 2018;Zhao et al., 2017).SOA formation capacity was enhanced by moth attacks on mountain birch (Rieksta et al., 2020;Yli-Pirilä et al., 2016) by up to a factor of 5 lasting several years (Taipale et al., 2021;Ylivinkka et al., 2020).The main increases from moth outbreaks came from SQT and GLV, similar to inductions from elevated O 3 in this study.Based on this, there is a potential that increased O 3 stress could lead to increased SOA formation in addition to plant damage.We found early leaf α-pinene emission rates to increase on average 15 times after exposure at 120 ppb compared to the control.A-pinene effectively fosters particle growth, indicating further SOA yield potential with O 3 exposure (Lee et al., 2006).
There are further implications of high O 3 concentrations in ambient air; BVOC compounds react faster in the atmosphere at high O 3 concentrations compared to low concentrations (Atkinson & Arey, 2003;Masui et al., 2023).This could impair plant-plant com- how elevated concentrations of acute O 3 exposure are affecting birch leaves that have not yet adapted to such conditions.There is also a knowledge gap in how mature birch trees respond to elevated O 3 exposure in situ and how well the trees recover in terms of BVOC emissions and photosynthetic rate after exposure.How mature birch trees respond to elevated O 3 at different leaf ages is also uncertain and can be of importance as the stress response can appear differently depending on leaf age, and a high O 3 level early in the growing season may reduce photosynthetic capacity at an early stage.To enhance our understanding of the responses of mature mountain birch trees in sub-Arctic areas to exposure to elevated O 3 , we defined the following aims: (1) investigate the effect of leaf age on BVOC emissions and photosynthetic rate, and (2) compare the potential difference in acute O 3 exposure effects on young leaves and older leaves.We also aimed to (3) quantify the effect of different acute O 3 exposure levels to identify thresholds in plant defense for carbon assimilation and BVOC emission, and (4) analyze the recovery of birch leaves after exposure to acutely increased O 3 concentrations.2 | ME THODS 2.1 | Site description Field measurements were done during the growing season of 2020 in the nature reserve at the Abisko Scientific Research Station (ANS) in the Swedish sub-Arctic (68°20′ N 19°02′ E) inside a birch forest dominated by mountain birch (Betula pubescens ssp.czerepanovii).
able photosynthesis system (LI-6400; LICOR, Lincoln, NE, USA), combined with an O 3 monitor (Model 202; 2B Technologies, Boulder, CO, USA) and an O 3 generator (Certizon C25; Erwin Sander Elektroapparatebau GmbH, Am Osterberg, Germany) connected with PTFE tubing (∅ 6.35 mm; Teflon, Swagelok, Solon, OH, USA).A schematic of the instrument setup, connections, and inlets and outlets are shown in Figure 2a.The pump provided ambient air to the system, passing the O 3 generator that was manually adjusted to different concentration levels (i.e.40, 80, or 120 ppb).The O 3enriched air was passed through a buffer volume of ~3 liters mixed with filtered 0 ppb O 3 air to enable stable concentrations.From the buffer volume, the air was led through a T-cross to both the LI-6400 leaf chamber and the O 3 monitor that was used to measure the O 3 concentration reaching the leaf chamber.An adjustable T-cross was also installed before the photosynthesis system for swapping between O 3 -enriched air and filtered 0 ppb O 3 air.When the 0 ppb O 3 measurements were ongoing, the T-crossing was directed to only have filtered 0 ppb O 3 air entering the chamber.When the O 3 exposure measurements started, this T-crossing was directed toward the O 3 -enriched air.Once the air passed the chamber head, it was directed to the O 3 monitor to assess the O 3 concentration the leaf was experiencing.
lowed an hour to acclimate to a leaf temperature of 20°C and photosynthetically active radiation (PAR) of 1000 μmol m −2 s −1 .The CO 2 concentration during the measurements varied with the ambient concentration, ranging from 350 to 430 ppm.BVOCs were sampled by drawing air through adsorbent tubes (Markes International Limited, Llantrisant, UK) packed with Tenax TA (a porous organic polymer) and Carbograph 1TD (graphitized carbon black) using flowcontrolled pocket pumps (Pocket Pump; SKC Ltd., Dorset, UK).To avoid O 3 oxidation of the BVOCs inside the tube, an O 3 filter (MnO 2 nets) was installed in front of the tube.The sampling time during the exposure phases was 60 min with a flow rate of 100 mL min −1 through the tubes, while the sampling time was 30 min with a flow rate of 200 mL min −1 during the control and recovery stages.The collected volume for each sample was 5 to 6 L. The net photosynthetic rate was measured simultaneously with the BVOC sampling using the LI-6400, herein only referred to as the photosynthetic rate.

(
TD; TurboMatrix 350, Perkin-Elmer) connected to a gas chromatograph (GC; Clarus 680, Perkin-Elmer) coupled to a mass spectrometer (MS; Clarus SQ 8 T, Perkin-Elmer).The samples were cryo-focused onto a dual-adsorbent cold trap (Tenax TA and Carbopack B) kept at −30°C.A DB-5 column (length 60 m, internal diameter (id.) 0.25 mm, F I G U R E 1 The measured (a) daily average air temperature (red line with daily maximum and minimum in a red-shaded area) and daily precipitation (blue bar) and (b) daily average incoming photosynthetic active radiation (PPFD; yellow line) for the growing season of 2020 at the Abisko Scientific Research Station.The gray-shaded areas indicate the time of the field visits for this study.Temperature and precipitation data were supplied by the Swedish Meteorological and Hydrological Institute (SMHI, 2023), and radiation data were supplied through the Abisko Scientific Research Station (ANS, 2023).
normplot, MATLAB 2022b, Mathworks, Inc., MA, USA), resulting in no normal distribution of the data.Any significant differences between the control leaves from the early and late summer were tested for leaf photosynthetic rate, stomatal conductance, transpiration rate, SLA, chlorophyll content, and the BVOC emission rates for unique compounds and the total emissions with a Kruskal-Wallis test (kruskalwallis, MATLAB R2022b; MathWorks, Inc., MA, USA) with a significance level set to p < .05.The significant differences for all leaves during the early or late summer between each step in the measurement sequence were also tested for photosynthetic rate, stomatal conductance, and the BVOC emission rates for unique compounds and total BVOC emissions, using two separate Kruskal-Wallis tests for each measurement time (early or late summer).After the Kruskal-Wallis test, a multiple comparisons procedure with Dunn-Sidák's approach (multcompare, MATLAB R2022b; MathWorks, Inc., MA, USA) was made to identify which groups were significantly different from each other, if any.Rough winds broke off some leaves during sampling; these leaves were removed from the data set and not analyzed.3| RE SULTS3.1 | BVOC emission patterns and photosynthetic rate from non-exposed (control) birch leavesThe first aim was to study differences depending on leaf age.Following the O 3 exposure sequence, mountain birch leaves (Betula pubescens ssp.czerepanovii) were measured starting at a concentration of 0 ppb to measure the constitutive BVOC emissions.The results show that early leaves had an average BVOC emission rate of 189 ± 189 ng g dw instead higher for the early leaves (OXY: 21 ± 51, ng g dw −1 h −1 SQT: 17 ± 25 ng g dw −1 h −1 , isoprene: 10 ± 16 ng g dw −1 h −1 ) compared to the late leaves (OXY: 5 ± 13 ng g dw −1 h −1 , SQT: 4 ± 4 ng g dw −1 h −1 , isoprene:F I G U R E 3The non-exposed (control) birch branches: (a) early summer BVOC emission (mean ± standard deviation (SD)) and photosynthetic rate (mean ± SD), (b) early summer emission blend (%), (c) late summer BVOC emission (mean ± SD) and photosynthetic rate (mean ± SD), and (d) late summer emission blend (%).The BVOC compounds are separated by color in their respective groups, with monoterpenes (MT) in red-pink, sesquiterpenes (SQT) in greens, and oxygenated compounds (OXY) in blues, where MBO is abbreviated from 2-methyl-3-buten-1-ol and AMCH from 4-acetyl-1-methylcyclohexene.Isoprene is separated on its own and colored yellow.5 ± 8 ng g dw −1 One of the aims was to investigate the extent to which the trees recovered after short-term O 3 exposure.The measurement sequence included three recovery phases where the leaves were exposed to 0 ppb after exposure to 40 ppb, 80 ppb, and 120 ppb O 3 concentrations.The birch trees showed different recovery patterns.Total emission rates from Birch 1 decreased for all recovery phases compared to the control for both early and late leaves.Birch 2 increased emission rates with each recovery phase for early leaves, while the emission rates for late leaves decreased during the recovery phases compared to the control state.The early leaves from Birch 3 indicated decreased emission rates for the first two recovery phases compared to the control, but increased to five times higher emission rates at the last recovery phase.The late leaves instead indicated small increases in emission rates during the recovery phases (Figure4; Table as described for the individual birches; early leaves had no large difference between control and 40 ppb, only increases of cis-3-hexenol F I G U R E 4 The recovery-phase birch branches' (a) early summer BVOC emission and photosynthetic rate during recovery phase 1, (b) early summer BVOC emission and photosynthetic rate during recovery phase 2, (c) early summer BVOC emission and photosynthetic rate during recovery phase 3, (d) late summer BVOC emission and photosynthetic rate during recovery phase 1, (e) late summer BVOC emission and photosynthetic rate during recovery phase 2, and (f) late summer BVOC emission and photosynthetic rare during recovery phase 3. The birch leaves were exposed to elevated O 3 at concentrations of 40 ppb, 80 ppb and 120 ppb prior to measuring the respective recovery phases 1-3 at an exposure concentration of 0 ppb.The standard deviation (SD) from the mean for the BVOC emission rates is

F
The O 3 exposure-phase birch branches' (a) early summer BVOC emission and photosynthetic rate at 40 ppb, (b) early summer BVOC emission and photosynthetic rate at 80 ppb, (c) early summer BVOC emission and photosynthetic rate at 120 ppb, (d) late summer BVOC emission and photosynthetic rate at 40 ppb, (e) late summer BVOC emission and photosynthetic rate at 80 ppb, and (f) late summer BVOC emission and photosynthetic rare at 120 ppb.The birch leaves were exposed to elevated O 3 at concentrations of 40 ppb, 80 ppb, and 120 ppb following a recovery phase of 0 ppb.
munications through BVOCs, which for the sub-Arctic area prone to moth outbreaks might have negative impacts as stress-induced signals would not reach far from the emitting plant.To conclude, future acute increases in tropospheric O 3 over the sub-Arctic can have large implications for mountain birch and their environment.Elevated O 3 was more stressful for early leaves, indicating a potential for lower stress thresholds in early summer, a season already experiencing higher levels of O 3 .If those levels increase more, it would cause plant stress, affecting the photosynthetic rate of the mountain birch and inhibiting plant growth.There were indications of leaf senescence in the early leaves after exposure to 120 ppb, further highlighting the negative impact of elevated O 3 .Late leaves were not as sensitive to the elevated O3 concentration in this study and could have better protection against O 3 stress, potentially due to a priming effect.However, stress-induced signals from SQT were seen from late leaves after exposure at 120 ppb.If elevated tropospheric O 3 becomes the norm in the sub-Arctic, it might have further implications for the local climate and is thus important to consider and take measures to prevent this from happening.